Decellularized Biomaterial from Non-Mammalian Tissue

ABSTRACT

The growth factor profile, connective tissue matrix constituents, and immunoprivileged status of urodele extracellular matrix (ECM) and accompanying cutaneous tissue, plus the presence of antimicrobial peptides there, render urodele-derived tissue an ideal source for biological scaffolds for xenotransplantation. In particular, a biological scaffold biomaterial can be obtained by a process that entails (A) obtaining a tissue sample from a urodele, where the tissue comprises ECM, inclusive of the basement membrane, and (B) subjecting the tissue sample to a decellularization process that maintains the structural and functional integrity of the extracellular matrix, by virtue of retaining its fibrous and non-fibrous proteins, glycoaminoglycans (GAGs) and proteoglycans, while removing sufficient cellular components of the sample to reduce or eliminate antigenicity and immunogenicity for xenograft purposes. The resultant urodele-derived biomaterial can be used to enhance restoration of skin homeostasis, to reduce the severity, duration and associated damage caused by post-surgical inflammation, and to promote progression of natural healing and regeneration processes. In addition, the biomaterial promotes the formation of remodeled tissue that is comparable in quality, function, and compliance to undamaged human tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) to U.S.provisional application 61/750,555, filed Jan. 9, 2013, the entirecontents of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

Tissue engineering efforts are ongoing to produce methods and materialsfor replacing biological functions, typically repairing or replacingwhole tissues or portions thereof In this regard, wound treatment andskin repair are areas of predominant focus, as the loss of skinintegrity due to illness or injury can lead to chronic, life threateningcomplications.

Wound healing involves complex interactions between cells, growthfactors, and extracellular matrix (ECM) components to reconstitutetissue following injury. The wound healing process in adult mammaliantissue has been well characterized and can be broken down into threestages—inflammation, proliferation, and remodeling.

Typically, in response to an incision or trauma the body conveys blood,blood products, and proteins into the void (also referred to as thecavity or negative space) formed at the wound. During earlyinflammation, a wound exudate begins to form under the influence ofinflammatory mediators and as a result of vasodilation. Fibrin andfibronectin present in clotting blood provide a scaffold over whichcells such as keratinocytes, platelets and leukocytes migrate to thewound site. Bacteria and debris are phagocytosed and removed, and growthfactors are released that stimulate the migration and division offibroblasts.

The subsequent stage of wound healing involves new tissue formation asfibrous connective tissue, termed granulation tissue (composed offibroblasts, macrophages and neovasculature) replaces the fibrin clot.New blood vessels are formed during this stage, and fibroblastsproliferate and produce a provisional ECM by excreting collagen andfibronectin. Nearly all mammalian cells require adhesion to a surface inorder to proliferate and function properly. The ECM fulfills thisfunction. Initially, the provisional ECM contains of a network of TypeIII collagen, a weaker form of collagen that is rapidly produced. Thisis later replaced by the stronger Type I collagen (which contributes toscar formation). At the same time, re-epithelialization of the epidermisoccurs. During this process, epithelial cells proliferate and migrateover the newly forming tissue as proteases such as metalloportineaes(MMPs) and collagenases at the leading edge of the migrating cells helpto invade the clot. These enzymes in addition to growth factor signaling(cell-cell interactions) and cell-ECM interactions (signal transductionfrom interactions between cells, integrins (cell surface receptors),laminin, collagen, fibronectin, and other ECM proteins) stimulate cellmigration into the wound and ECM degradation.

Finally, in the remodeling phase, collagen is remodeled and realignedalong tension lines and cells that are no longer needed are removed byapoptosis. Wound contraction occurs as fibroblasts transform intomyofibroblasts through their interactions with ECM proteins and growthfactors. Myofibroblasts then interact with collagen, vitronectin, andother ECM proteins to contract the wound. As the remodeling phaseproceeds, fibronectin and hyaluronic acid are replaced by collagenbundles that lend strength to the tissue.

By applying biological, chemical and engineering principles to tissuerepair and regeneration, tissue engineers have developed transplantableproducts for use in promoting the tissue repair and regenerationprocesses. The ability to restore biomechanical function of damagedtissue presents a true challenge. In response, both synthetic andbiological scaffold products have been developed that mimic(to someextent) tissue structure and mechanical behavior to promote tissuerepair. Such products serve as a temporary replacement, bothmechanically and functionally, for damaged, diseased or absent tissue.

Ideally, transplantable scaffold products should support cell adhesion,proliferation and differentiation and act as an interim syntheticextracellular matrix (ECM) for cells prior to the formation of newtissue. Scaffold materials should be biocompatible, biodegradable andexhibit low antigenicity. The implant should degrade at a rate roughlyequal to that of the new tissue formation. Once implanted, the scaffoldmust have the mechanical properties necessary to temporarily offerstructural support until the new tissue has formed. Additionally,scaffold products must be porous, providing an appropriate path fornutrient transmission and tissue ingrowth. Tissue scaffolds also shouldpromote fast healing and facilitate the development or regeneration ofnew tissue that resembles normal host tissue in both appearance andfunction. To this end, implanted scaffold products should offer (i)bioactive stimulation, e.g., protein and molecular signaling, toencourage cell migration, proliferation and differentiation, and (ii)mechanical or structural support for these processes.

Today, the development of synthetic scaffolds is an area of activeresearch. Synthetic scaffolds have been manufactured from chemicalcompounds such as fibrous polymers, gelatin, apatite, andpolymer/ceramic composites, polylactic acid, collagen. These scaffoldsare designed to mimic the structure of the naturally occurring ECM andhave shown some success in bone tissue engineering.

In addition to synthetic scaffold products, biological scaffoldsobtained from mammalian tissues are commercially available for use asallografts (transplanted cell or tissue from a donor of the samespecies) or xenografts (transplanted cells or tissue from a donor of adifferent species). Biological scaffolds are composed of mammalian ECMharvested from, for example, dermis, urinary bladder, small intestine,mesothelium, pericardium, bone or aminiotic membrane of various mammalsincluding human (either live donor or cadaver), porcine, bovine andequine. These commercially available products are commonly used for therepair and reconstruction of injured or missing tissues and organs suchas soft tissue, tendons, cardiac tissue, neural tissue, chronic wounds,dura mater, bone and cornea.

Biological scaffold products may comprise skin cells in addition toextracellular matrices produced by tissue and subjected to adecellularization process. They are contacted with a wound site to givemechanical support for cell migration and proliferation as part of thewound healing process. In addition, factors such as growth factors orother proteins also may be provided that promote the wound healingprocess. The mechanical and material properties of biological scaffoldsand the host tissue response to these biomaterials are believed to beinfluenced by the three dimensional configuration of the material andproduction processing methods. It further is believed that growthfactors, surface topology and the distribution of surface ligands andmodulation of the host innate immune response all contribute to theeventual functional performance of biological scaffolds for tissuerepair or reconstruction. Tottey et al., Biomaterials 32: 128-36 (2011).

In transplantation the use of human amniotic membrane (AM) hasparticular advantages, due to the structure of the relatively thickbasement membrane, associated devitalized amniotic epithelial cells andstroma, and corresponding growth factor profile and structural proteincomposition. Meller et al., Dtsch Arztebl Int'l 108: 243-8 (2011). Forexample, AM contains epidermal growth factor (EGF) and keratinocytegrowth factor (KGF), which are important growth factors for promotingwound healing. In addition, laminin and type VII collagen present in theAM elicit an epitheliotropic effect. AM also is thought to reduce theexpression of various growth factors and pro-inflammatory cytokineswhile releasing anti-inflammatory cytokines such as IL-10, IL-1 receptorantagonists, thus modulating the inflammatory response favorably forwound healing. AM is immunoprivileged, moreover, likely by virtue of lowMHC I expression, and so rejection of AM tissue is uncommon. Thesecharacteristics make AM an ideal substrate, for instance, with respectto ocular surface reconstruction, in pelvic reconstruction, and in thetreatment of ulcers, among other wound-healing applications.

The use of conventional tissue scaffold products is not withoutdrawbacks, however. Tissue harvesting from human donors can produceundesirable consequences such as donor site morbidity or infectionassociated with removal of skin for donation. Disease transmission riskand intersample variation are additional drawbacks associated withbiological scaffold products. In addition, it may be difficult to obtainsufficient tissue components necessary to cover large areas of damagedtissue. Furthermore, conventional biological and synthetic materials canbe costly, not effective in many instances, and limited in availability.

Accordingly, an abiding exists need for suitable tissue substratebiomaterial for use in transplantation to promote tissue regenerationwhile restoring functionality. Both the research industry and themedical transplant community would benefit from such a product that isreadily available, does not impose additional complications to a donoror recipient (such as requiring an additional surgery to harvest thesubstrate), and exhibits all or some of the inherent materialfunctionality reflective of the physiochemical, electrochemical, andbiochemical properties of natural tissue.

SUMMARY OF THE INVENTION

The biomaterial of the present invention is obtained from tissue of aurodele. “Urodele” here denotes a salamander of the order Urodela, alsoknown as the order Caudata, in the class Amphibia. In terms of phylogenythe relevant families include Ambystomatidae, Cryptobranchidae,Amphiumidae, Proteidae, and Sirenidae. See Wiens et al., Syst. Biol. 54:91-110 (2005), the contents of which are incorporated here by referencein their entirety. Accordingly, the urodele category includes, forexample, the Pacific Giant Salamander (Andrias davidianus), the TigerSalamander (Ambystoma tigrinum), and the Mexican Axolotl (Ambystomamexicanum). The present inventor has recognized that the skin and ECM ofurodeles possess desirable characteristics analogous to those of AM,making them an ideal source of biomaterial for xenotransplantation.

Urodele ECM and Tissue Regeneration

As amphibians, most urodeles begin life as aquatic animals in a larvalstate and undergo metamorphosis from a juvenile form with gills to anadult, terrestrial, air-breathing form with lungs. During metamorphosis,a urodele's physical features are altered in preparation for life onland. These alterations include caudal fin resorbtion, thickening of theskin, the development of dermal glands and resorption of gills. Sexualmaturity also occurs during this time in most urodeles. Some families ofurodeles are “neotenic,” which means that individuals with such familiescan exhibit juvenile features, such as gills and fins, even afterreaching sexually maturity. Indeed, neotenic urodeles often retain theiraquatic (juvenile) form for the duration of their lives. Thus, theMexican Axolotl normally remains in the neotenic state throughout itsadult life although, under certain circumstances, it can undergometamorphosis and transform into a terrestrial form.

Axolotls also are known for their ability to regenerate amputated bodyparts, which typically results in the complete restoration of both thestructure and function of the damaged limb or organ. Aquatic axolotlsundergo rapid re-epithelialization during wound healing and limbregeneration, both of which are scar-less processes. Similarly,metamorphic terrestrial axolotls retain several larval skin features andalso exhibit scar-free wound healing, albeit at a slower rate than theiraquatic, pre-metamorphic counterpart.

The healing process in axolotls varies from that observed in adultmammals. The axolotl process more closely resembles the scar-freehealing process of fetal and embryonic wounds. Thus, such woundslikewise exhibit re-epithelialization and basement membrane reformationthat occur at a faster rate (is “enhanced”) than do the correspondingevents in postnatal mammals.

Moreover, the cutaneous and subcutaneous structures of an axolotlresemble that of the amniotic/chorionic interface, in the sense thataxolotl skin is composed of fused ectoderm and mesoderm. Axolotl skinalso is rich in growth factors and antimicrobial peptides, similar tothe AM. Furthermore, axolotl ECM is immunoprivileged and containscollagen III and tissue inhibitors of metalloproteinases (TIMPs), interalia, also in resemblance to AM.

Of particular importance for transplantation purposes, more generally,is the immunologically privileged state of the human neonate (e.g.,fetal dermis) and the AM, a state mirrored by axolotl ECM, wherebyimmogenicity is rarely manifested. By virtue of the reduced immuneresponse and the generally decreased inflammatory response as comparedto adult humans, neonatal and axolotl skin healing alike are notcharacterized by accelerated tissue resorption, as is observed in adulthuman wound healing. Rather, the growth factor profile, enzymaticactivity, structural composition and immunomodulating effect of urodeleand neonate tissues alike favor an appropriately staged removal ofstructural scaffolding and tissue growth into the resulting negativevoid space, in addition to enhanced re-epithelialization, during woundhealing. This results in an optimal wound healing environment andprocess. Also, the high concentration of antimicrobial peptides presentin AM and urodele tissue further contributes to the favorableenvironment and enhanced re-epithelialization observed during woundhealing.

A key aspect of the present invention is the inventor's recognition thatthe growth factor profile, connective tissue matrix constituents, andimmunoprivileged status of urodele ECM and accompanying cutaneoustissue, plus the presence of antimicrobial peptides therein, renderurodele-derived tissue an ideal source for biological scaffolds forxenotransplantation.

In accordance with the invention, therefore, a biological scaffoldbiomaterial is provided that is the product of a process comprising (A)obtaining a tissue sample from a urodele, where the tissue comprisesECM, inclusive of the basement membrane, and (B) subjecting the tissuesample to a decellularization process that maintains the structural andfunctional integrity of the extracellular matrix, by virtue of retainingits fibrous and non-fibrous proteins, glycoaminoglycans (GAGs) andproteoglycans, while removing sufficient cellular components of thesample to reduce or eliminate antigenicity and immunogenicity forxenograft purposes. Also provided is methodology for using theurodele-derived biomaterial to enhance restoration of skin homeostasis,to reduce the severity, duration and associated damage caused bypost-surgical inflammation, and to promote progression of naturalhealing and regeneration processes. In addition, biomaterial of theinvention promotes the formation of remodeled tissue that is comparablein quality, function and compliance to undamaged human tissue.

Decellularization

The biomaterial of the invention is produced by decellularizing a tissuesample obtained from a urodele. The primary constituent of the resultingurodele biomaterial is ECM, possibly with devitalized epithelial cells,which can retain moisture and otherwise protect the wound-healingenvironment.

Urodele skin is one example of an appropriate starting material for thepresent invention. Thus, the starting material that is subjected todecellularization can comprise urodele dermis and basement membrane,with or without epidermis. Even upon decellularization, moreover, thebiomaterial of the invention can comprise, with the ECM, adjacentepithelial cells that may be rendered non-viable by the process.Alternatively, non-cutaneous urodele tissues can serve as the startingmaterial of the invention, particularly those comprising a basementmembrane or epithelial tissues that form the lining of various bodycavities, i.e., parietal mesothelial tissues found, for example in thethoracic cavity, the abdominal cavity, and pericardium. Tissues thatcontain substantial amounts of fibrous connective tissue, such ascartilage, tendon, bone, dura mater and fascia, also are illustrative ofappropriate starting materials of the present invention.

Effected via any conventional decellularization methodology, urodeletissue decellularization is performed to remove immunogenic cellularantigens that can induce an inflammatory response or immune-mediatedtissue rejection, while preserving the structural integrity andcomposition of the associated ECM. Generally, ECM structural components,many if not all of which remain intact following decellularization, arewell-tolerated by xenogeneic recipients. ECM components that may bepresent in the final biomaterial of the invention include proteins suchas collagen (e.g., fibrous collagen I and collagen III, as wellnon-fibrous collagen IV, collagen V and collagen VII), elastin,fibronectin, laminin, vitronectin, thrombosponsdins, osteopontin andtenascins, plus GAGs (e.g.,the proteoglycans, decoran and versican andsulfated GAGs, e.g., heparin sulfate, keratan sulfate, dermatan sulfateand chondroitin sulfate) and growth factors such VEGF, BMP, TGF and FGF.For some indications the post-decellularization material comprises atleast collagen IV, laminin, sulfated GAGs and one or more growth factorsin amounts that approximate pre-decellularization levels when viewed viahistological and immunohistological staining.

Suitable techniques for decellularizing tissues, pursuant to theinvention, include physical methods such as freezing, direct pressureapplication, sonication, and agitation. In addition or in thealternative, chemical methods can be employed, such as alkaline and acidtreatments, application of detergents (including amphoteric, cationic,anionic and non-ionic detergents), organic solvents, hypotonic orhypertonic solutions and chelating agents. Enzymatic approachesincluding protease digestion and treatment with one or more nucleasesalso may be used to decellularize urodele tissue. In addition oralternatively, the urodele tissue is subjected to cleaning,sterilization, disinfection, antibiotic treatment and/or viralinactivation.

According to one aspect of the invention, a biomaterial is provided. Thematerial is produced by the process that includes (A) obtaining a tissuesample from a urodele, which tissue sample comprises extracellularmatrix, and (B) decellularizing the sample to retain structural andfunctional integrity while removing sufficient cellular components ofthe sample to reduce or eliminate antigenicity of the biomaterial as axenograft. In some embodiments, decellularizing comprises subjectingsaid tissue sample to an alkaline treatment. In embodiments, the processcan further comprise subjecting said sample to sterilization. Inembodiments, the process can further comprise devitalizing cells.

According to one aspect of the invention, a tissue graft is provided.The graft includes extracellular matrix components derived from aurodele. In embodiments, the extracellular matrix components aresubstantially free of components that induce an immune response whenimplanted as a xenograft. In embodiments, the extracellular matrixcomponents are non-toxic.

According to one aspect of the invention, a decellularized Urodele ECMis provided. In any of the embodiments, the decellularized ECM can bederived from Axolotl tissue. In any of the embodiments, thedecellularized ECM can include basement membrane. In any of theembodiments, the decellularized ECM can be infused with, coated with,combined with or attached, covalently or non-coalently, to an agentxenogenic to a Urodele. In any of the embodiments, the agent can be anyone or any combination of a growth factor, a cytokine, a chemokine, aprotein, a carbohydrate, a sugar, a steroid, an antimicrobial agent, asynthetic polymer, an adhesive, a drug and/or a human agent (i.e., anagent found in a human, isolated, synthetically or recombinantlyproduced). Further such agents forming part of the invention aredescribed in more detail below. In some embodiments, the agent is acell, optionally a human cell. In some embodiments, the agent is aprogenitor cell, optionally a human progenitor cell. Further such cellsforming part of the invention are described in more detail below. In anyof the embodiments, the decellularized ECM can take on any variety ofshapes, as the material can be formed, laminated, homogenized, gelled,etc. In some embodiments, the ECM is a sheet. The sheet optionally caninclude perforations. The sheet optionally can include a backing and/oran adhesive. The backing may be biodegradable or may benon-biodegradable. In some embodiments, the decelularized ECM is a drypowder. In some embodiments the decelularized ECM is a reconstitutedgel. In any of the embodiments, the ECM can be sterile.

According to one aspect of the invention, a package is provided. Thepackage contains sterile, decellularized Urodele ECM or a Urodelefraction derived from the sterile, decellularized Urodele ECM. Thepackage can contain any of the ECMs described above. For example, thepackage can include a sheet, a dry powder or a reconstituted gel ofdecellularized Urodele ECM. The package can contain any product, forexample any implant, that comprises sterile, decellularized Urodele ECMor a Urodele fraction derived from the sterile, decellularized UrodeleECM. Such an implant may be made in whole or only in minor part of theECM of the invention.

The invention also provides a sterile medical implant comprisingdecellularized Urodele ECM or a Urodele fraction derived from thedecellularized Urodele ECM. Examples of such medical implants include abiocompatible sheet, mesh, gel, graft, plug, tissue or device. Devicesinclude, for example, coated stents, bone replacements, jointreplacements, implantable hardware and the like. The implant can befabricated entirely or in part from the ECM. The implant also canencapsulate, can be infused with, coated with, impregnated with,laminated with, or covalently or non-covalently attached to the ECM ofthe invention.

The invention also provides a material, the material being coated with,impregnated with, encapsulating, or having attached thereto isolated,decellularized Urodele ECM or a Urodele fraction derived from theisolated, decellularized Urodele ECM. The material can be natural orsynthetic. Examples are metals, plastics, ceramics and fibers.

According to one aspect of the invention, a tissue culture system isprovided. The system comprises (a) an isolated Urodele decellularizedECM, (b) tissue culture medium, and (c) cells xenogenic to the Urodele.The cells may be from animal, and in some embodiments, the cell is amammalian cell. The cell can be any type of cell capable of culture. Inembodiments, the cell is a human cell, optionally a progenitor cell.

According to one aspect of the invention, a conditioned tissue culturemedium is provided. The medium, which can be any commonly used in liquidtissue culture, is conditioned with isolated Urodele decellularized ECMor a Urodele fraction derived from isolated, decellularized Urodele ECM.Numerous liquid tissue culture media are commercially available and wellknown to those of ordinary skill in the art.

According to one aspect of the invention, a device is provided. Thedevice is at least two sheets of isolated Urodele decellularized ECMlaminated to one another. The sheets can be from the same or differenttissue. The sheets can an orientation and can be oriented in the samedirection or oriented at angles to one another. The sheets can furthercomprise any agent xenogenic to the Urodele, which agent may be coatedon, infused or impregnated within, or otherwise attached to one or moreof the laminated sheets.

In any embodiment described above involving a sheet, the sheet mayfurther comprise a backing and/or adhesive.

According to one aspect of the invention, a product is provided. Theproduct is prepared by isolating Urodele ECM from a Urodele,decellularizing the ECM, and sterilizing the decellularized ECM. Thepreparation of the device can further involve any one or more of thefollowing steps (presented in no particular order): forming the ECM intoa shape, homogenizing the ECM, laminating the ECM to a material,combining agents with the ECM such as by coating, impregnating, orotherwise attaching the agent to the ECM, and so on.

According to one aspect of the invention, a method of preparing abiologic material is provided. The method involves (A) obtaining atissue sample from a urodele, which tissue sample comprisesextracellular matrix, and (B) decellularizing the sample to removesufficient cellular components of the sample to reduce or eliminateantigenicity of the biomaterial as a xenograft. In embodiments, themethod can further involve performing the decellularization in a mannerto retain structural and functional integrity of the ECM sufficient topermit the ECM to be useful as a matrix upon and within which cells cangrow. In any of the embodiments, the method can further involvehomogenizing the ECM to form a particulate or powder. In someembodiments, the method can further involve reconstituting the powder asa gel. In any of the embodiments, the method can further involvesterilizing the ECM. In any of the embodiments, the method can furtherinvolve attaching the ECM to an agent xenogenic to a Urodele.

According to another aspect of the invention, the materials such asimplants, devices, sheets, gels and powders can be used in methods fortreating subjects, where the materials are applied to wounds, surgicalbeds, and to internal and external tissues, generally, to preventadhesion, provide tissue support, for example for suturing tissue, fortreating a hernia or as a tissue plug, for treating burns andderm-abrasion, as well as other conditions described below.

In any of the embodiments above, the ECM is or can be isolated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows native axolotl tissue samples prepared for histologicalexamination, to identify EC elements.

FIG. 2 hematoxylin and eosin (H&E) and Alcian Blue staining of nativeaxolotl dermal tissue and human amniotic membrane (40× magnification).

FIG. 3 shows immunohistocemical staining via species-specific collagenIV and laminin antibodies of native axolotl dermal tissue and humanamniotic membrane tissue, at 40× magnification.

FIG. 4 depicts axolotl skin and human amniotic membrane samples preparedfor histological examination, to identify EC elements.

FIG. 5 illustrates a histological evaluation of H&E-stained, pairednative and post-processed sections of axolotl dermal tissue.

FIG. 6 presents ELISA data for DNA content in pre- and post-processedaxotol tissue, with comparison data from human amniotic membrane tissue.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

“Antimicrobial polypeptides” (or “AMPs”) means small peptides ofvariable length, sequence and structure with broad spectrum activityagainst a wide range of microorganisms including bacteria, viruses,fungi, protozoa, parasites, prions, and tumor/cancer cells. (See, e.g.,Zaiou, J Mol Med, 2007; 85:317; herein incorporated by reference in itsentirety). AMPs have broad-spectrum of rapid onset of killingactivities, with potentially low levels of induced resistance andconcomitant broad anti-inflammatory effects. Anti-microbial polypeptidesinclude defensins, such as a-defensins (e.g., neutrophil defensin 1,defensin alpha 1, neutrophil defensin 3, neutrophil defensin 4, defensin5, defensin 6), β-defensins (e.g., beta-defensin 1, beta-defensin 2,beta-defensin 103, beta-defensin 107, beta-defensin 110, beta-defensin136), and θ-defensins. Anti-microbial polypeptides include cathelicidinssuch as hCAP18.

“Biocompatible” means that a composition and its normal degradationproducts in vivo are substantially non-toxic and non-carcinogenic in asubject within useful, practical and/or acceptable tolerances.

“Cytocompatible” means that a composition can sustain the viability andgrowth of a population of cells.

“Decellularized ECM” means extra cellular matrix sufficiently free ofcellular components to eliminate or reduce antigenicity of the extracellular matrix to an extent where the matrix would be considerednon-toxic as a xenograft.

“Isolated” when used in connection with the ECM of the invention meansseparated from other Urodele tissue.

“Non-toxic” means that a composition, when implanted in a subject,causes little or no adverse reaction or substantial harm to cells andtissues in the body, and does not cause a substantial adverse reactionor substantial harm to cells and tissues in the body, for instance, thecomposition does not cause necrosis, an infection, or a substantialimmune response resulting in harm to tissues from the implanted orapplied composition.

“Progenitor cell” means a cell that can differentiate under certainconditions into a more-differentiated cell type. Non-limiting examplesof progenitor cells include stem cells that may be totipotent,pluripotent, multipotent stem cells, or referred to as progenitor cells.Additional non-limiting examples of progenitor cells includeperivascular stem cells, blastema cells, and multilineage progenitorcells.

“Retain structural and functional integrity” used in connection with theECM of the invention means retaining sufficient structure and functionto permit and support the use of the matrix as a substrate for thegrowth of cells in vivo or in vitro.

“Subject” means an animal. In some embodiments the animal is a mammal.The mammal can be a dog, cat, a horse, a cow, a goat, a sheep, a pig ora non-human primate. In any embodiment the mammal can be a human.

“Treatment” or “treating” means administration or application to asubject by any suitable dosage means, regimen and route of a compositionwith the object of achieving a desirable clinical/medical end-point,such as assisting in wound healing, tissue closure, bulking tissue,preventing tissue adhesion, providing structural support to tissue,providing a protective barrier, correcting a defect, etc.

“Urodele fraction derived from decellularized Urodele ECM” means anextract or isolate of decellularized Urodele ECM maintaining sufficientcharacteristics of a Urodele in terms of chemical structure and/orrelative chemical concentrations of two (or three, or four, or five ormore) chemical entities in the extract or isolate to distinguish theextract as obtained from a Urodele by any one or more of electronmicroscopy, HPLC, immunohistochemistry, and the like.

General Preparative Methodology

According to the invention, urodele tissue samples obtained fordecellularization can be treated in the manner detailed in US2008/0046095 or US 2010/0104539. Thus, tissue samples may be subjectedto cleaning and chemical decontamination. In this manner, a tissuesample is washed for approximately 10 to 30 minutes in a sterile basincontaining 18% NaCl (hyperisotonic saline) solution that is at or nearroom temperature. Visible cellular debris, such as epithelial cellsadjacent to the tissue basement membrane, is gently scrubbed away usinga sterile sponge to expose the basement membrane. Using a bluntinstrument, a cell scraper or sterile gauze, any residual debris orcontamination also is removed. Other techniques including, but notlimited to, freezing the membrane, physical removal using a cellscraper, or exposing the cells to nonionic detergents, anionicdetergents, and nucleases also may be used to remove cells.

In one embodiment, urodele tissue is decellularized using alkalinetreatment.

The tissue is placed into a sterile container, such as a Nalgene jar,for the next step of chemical decontamination. Thus, each container isaseptically filled with 18% saline solution and sealed (or closed with atop). The containers then are placed on a rocker platform and agitatedfor between 30 and 90 minutes, which further cleans the tissue ofcontaminants.

In a sterile environment using sterile forceps, the tissue is gentlyremoved from the container containing the 18% hyperisotonic salinesolution and placed into an empty container. This empty container withthe tissue is then aseptically filled with a pre-mixed antibioticsolution. Preferably, the premixed antibiotic solution is comprised of acocktail of antibiotics, such as Streptomycin Sulfate and GentamicinSulfate. Other antibiotics, such as Polymixin B Sulfate and Bacitracin,or similar antibiotics available now or in the future, are suitable aswell. It is preferred that the antibiotic solution be at roomtemperature when added so that it does not change the temperature of orotherwise damage the tissue. This container containing the tissue andantibiotics is then sealed or closed and placed on a rocker platform andagitated for, preferably, between 60 and 90 minutes. Such rocking oragitation of the tissue within the antibiotic solution further cleansthe tissue of contaminants and bacteria.

In a sterile environment, the container is opened and, using sterileforceps, the tissue is gently removed and placed in a sterile basincontaining sterile water or normal saline (0.9% saline solution). Thetissue is allowed to soak in place in the sterile water/normal salinesolution for at least 10 to 15 minutes. The tissue may be slightlyagitated to facilitate removal of the antibiotic solution and any othercontaminants from the tissue.

In some cases, the present invention involves treating urodele tissueusing a chemical sterilization methodology, as illustrated theTutaplast® and Allowash® procedures, optionally in combination withmechanical processes that gently agitate chemical agents, as in theBioCleanse® system. Thus, urodele tissue is subjected to oxidativeand/or alkaline treatments as well as osmotic treatment to break downcell walls, to inactivate pathogens, and to remove bacteria. Inaddition, tissue may be subjected to delipidization, solvent dehydration(to permit room temperature storage of tissue without damaging thecollagen structure) and/or low-dose gamma irradiation to ensuresterility of the final product.

Efficient cell removal upon decellularization can be verified by variousknown means, including histological analyses to detect nuclear andcytoplasmic structures, immunohistochemical or immunofluorescentassaying for indicative intracellular proteins, and DNA detection. Thenature of desirable components in the final urodele-derived scaffoldbiomaterial varies depending on the clinical indication being treated.Once a particular indication is identified, the knowledgeable cliniciancan determine which components in the urodele tissue sample should beretained in the final scaffold product, and standard methodology can beemployed to ensure that the desired components are present followingdecellularization.

Samples may be viewed histologically before, during, and/or afterdecellularization to monitor the process and to confirm that the desireddegree of cellular component removal is reached. For instance, tissuescan be analyzed for cytoskeletal content to gauge sufficientdecellularization. Intracellular protein content also may be assayed todetermine if decellularization is sufficient. In addition, the tissuesample thickness and chemical makeup may be monitored to determine whensufficient decellularization has been achieved. Periodic monitoringduring processing allows for a real time response to the observed tissueproperties.

In some cases, a sufficiently decellularized tissue comprises no morethan 50 ng dsDNA per mg ECM dry weight. Alternatively, for someindications, a sufficiently decellularized tissue lacks visible nuclearmaterial in a tissue section stained with 4′,6-diamindino-2-phenylindole(DAPI) or haematoxyilin and eosin (H&E).

In scenarios where removal of an adjacent epithelial cell layer isrequired, the presence or absence of epithelial cells remaining in thesample can be evaluated using techniques known in the art. For example,after removal of the epithelial cell layer, a representative tissuesample from the processing lot is placed onto a standard microscopeexamination slide. The tissue sample is then stained using Eosin Y Stainand evaluated as described below. The sample is then covered and allowedto stand. Once an adequate amount of time has passed to allow forstaining, visual observation is done under magnification. The presenceof cells and cellular material will appear darker than the areas whichhave been de-epithelialized.

Once cellular removal has progressed sufficiently, conventional methodsare employed to confirm the retention of desired structural andfunctional properties of the remaining ECM scaffold. The specificstructural testing that should be conducted depends on the intendedclinical application of the final scaffold product. In some cases, theurodele tissue starting material may be monitored before, during, andafter decellularization to ensure that the desired structural componentsand configuration are maintained in the final product.

One method for determining whether the desired ECM components arepresent involves staining parallel tissue sections and examining themhistologically to determine whether the desired constituents andstructural orientation of the urodele tissue have been preserved. Forinstance, urodele tissue can be stained with H&E and immunoperoxidasestain for laminin to assess preservation of ECM and laminin. In general,the three-dimensional configuration of ECM components remaining in thefinal biomaterial scaffold product should approximate that ofpre-decellularized material when viewed via histological staining.Another component one can assay for is AMPs, as the ECM of the inventionis rich in AMPs.

Accordingly, the urodele-derived biomaterial of the invention comprisesECM components useful for directing enhanced re-epithelialization andpromoting efficient tissue regeneration or wound healing. The inventivebiomaterial also serves as a matrix and reservoir for bioactive peptidessuch as growth factors, adhesion proteins and AMPs. Accordingly, thebiomaterial functions effectively as a biological scaffold for tissueregeneration, providing both the necessary bioactive stimulation andstructural support. The product can be used as is, cut into smallerpieces or shapes, laminated to itself or other materials, pre-puncturedto provide openings for securing attachments, formed into desired threedimensional shapes, as well as other formats, discussed in more detailbelow.

Powders and Gels

In embodiments, the scaffold can be further processed into small grainsor a powder. The fine particles can be hydrated in water, saline or asuitable buffer or medium to produce a paste or gel. This fine material,paste or gel produced from it may be used for a multitude of purposes,described in greater detail below.

Although numerous methods exist, two exemplary methods may be used toproduce a particulate form of the scaffold. The first method involvedlyophilizing the disinfected material and then immersing the sample inliquid nitrogen. The snap frozen material is then reduced to smallpieces with a blender so that the particles are small enough to beplaced in a rotary knife mill, such as a Wiley mill. A #60 screen can beused to restrict the collected powder size to a desired size, forexample less than 250 mm. A Sonic sifter or other classification devicecan be used to remove larger particles and/or to obtain a particle sizedistribution within a desired range. A second method is similar to theprevious method except the sample is first soaked in a 30% (w/v) NaClsolution for 5 min. The material is then snap frozen in liquid nitrogento precipitate salt crystals, and lyophilized to remove residual water.This material is then comminuted as described in above. By precipitatingNaCl within the sample, it is expected that the embedded salt crystalswould cause the material to fracture into more uniformly sizedparticles. The particles are then suspended in deionized water andcentrifuged for 5 min at 1000 rpm three times to remove the NaCl. Thesuspension is snap frozen and lyophilized again. Finally, the powder isplaced in a rotary knife mill to disaggregate the individual particles.

The powder can be hydrated to create a gel, with or without othergelling materials to supplement gelling.

The powder, paste or gel can be applied without further processing totreat a subject. It can be sprayed, painted, injected or otherwiseapplied to a wound or surgical site. The gel can be shaped. The powder,paste or gel also can be placed inside a “bag”, such as a polymericsynthetic material or a ECM sheet as described herein to produce alarger three-dimensional structure, such as an orthopedic implant forcartilage repair (e.g., knee or TMJ cartilage repair) or an implant forbreast reconstruction or augmentation. In such a case, a bag of adesirable size and shape is formed from sheets of ECM material or otherbiocompatible polymeric material, and then the bag or cover can befilled with the tissue-derived powder or gel described herein. The shapeof the device or implant can vary with its intended use. The bag may bemolded into a useful shape by any useful molding technique, such as theshape of cartilage for the ear, nose, knee, TMJ, rib, etc., prior tofilling the molded bag with the scaffold material described herein. Inone example, a biodegradeable polymeric matrix (e.g., PEUU or PEEUU) issprayed or electrodeposited onto a mold. The resultant molded cover canthen be filled with the material. Heat, for example, may be used to sealthe cover.

Additives

In another embodiment, at least one agent xenogenic to a Uroldele isadded to the ECM or Urodele fraction thereof before it is implanted inthe subject, otherwise administered to the subject or used in cellculture. Generally, the agents include any agent useful in cell cultureor as a therapeutic or therapeutic adjuvant. The agents can be coatedon, infused into or otherwise covalently or non-covalently attached toor incorporated onto or into the ECM of the invention. The agents alsocan be otherwise combined with a product that contains the ECM, forexample, as by mixing powders of the agent and ECM together. Each agentmay be used alone with the ECM of the invention or in combination withother agents. Non-limiting examples of such agents include antimicrobialagents, growth factors, cytokines, chemokines, emollients, retinoids,steroids, and cells, including but not limited to the subject's owncells.

In certain non-limiting embodiments, the agent is a growth factor.Non-limiting examples of growth factors include basic fibroblast growthfactor (bFGF), acidic fibroblast growth factor (aFGF), vascularendothelial growth factor (VEGF), hepatocyte growth factor (HGF),insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), platelet derivedgrowth factor (PDGF), stromal derived factor 1 alpha (SDF-1 alpha),nerve growth factor (NGF), ciliary neurotrophic factor (CNTF),neurotrophin-3, neurotrophin-4, neurotrophin-5, pleiotrophin protein(neurite growth-promoting factor 1), midkine protein (neuritegrowth-promoting factor 2), brain-derived neurotrophic factor (BDNF),tumor angiogenesis factor (TAF),corticotrophin releasing factor (CRF),transforming growth factors .alpha. and .beta. (TGF-.alpha. andTGF-.beta.), interleukin-8 (IL-8), granulocyte-macrophage colonystimulating factor (GM-CSF), interleukins, and interferons. Commercialpreparations of various growth factors, including neurotrophic andangiogenic factors, are available from R & D Systems, Minneapolis,Minn.; Biovision, Inc, Mountain View, Calif.; ProSpec-Tany TechnoGeneLtd., Rehovot, Israel; and Cell Sciences®, Canton, Mass.

In certain non-limiting embodiments, the therapeutic agent is anantimicrobial agent, such as, without limitation, an anti-microbialpeptide, isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine,rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin,azithromycin, clarithromycin, dapsone, tetracycline, erythromycin,ciprofloxacin, doxycycline, ampicillin, amphotericin B, ketoconazole,fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin,pentamidine, atovaquone, paromomycin, diclazaril, acyclovir,trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir,iatroconazole, miconazole, Zn-pyrithione, and silver salts such aschloride, bromide, iodide and periodate.

In certain non-limiting embodiments, the therapeutic agent is ananti-inflammatory agent, such as, without limitation, an NSAID, such assalicylic acid, indomethacin, sodium indomethacin trihydrate,salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal,diclofenac, indoprofen, sodium salicylamide; an anti-inflammatorycytokine; an anti-inflammatory protein; a steroidal anti-inflammatoryagent; or an anti-clotting agents, such as heparin.

Other drugs that may promote wound healing and/or tissue regenerationmay also be included.

The agent may be dispersed within the scaffold by any useful method,e.g., by adsorption and/or absorption. For example, the therapeuticagent may be dissolved in a solvent (e.g., DMSO) and added to thescaffolding. In another embodiment, the agent is mixed with a carrierpolymer (e.g., polylactic-glycolic acid microparticles, agarose, apoly(ester urethane) urea elastomer (PEUU) or a poly(ether esterurethane) urea elastomer (PEEUU)), which is subsequently dispersedwithin or applied to the scaffold. By blending the agent with a carrierpolymer or elastomeric polymer, the rate of release of the therapeuticagent may be controlled by the rate of polymer degradation and/or byrelease from the polymer by diffusion or otherwise. Likewise, atherapeutic agent may be provided in any dissolvable matrix for extendedrelease, as are known in the pharmaceutical arts, including sugar orpolysaccharide matrices. The agent also may be included with thepowdered ECM and gelled with the powdered ECM. The agent may becovalently attached to the ECM of the invention. The foregoing are meantto be non-limiting examples.

Extracts

In addition to the decellularized ECM in its native state or ground as aparticulate or powder, the invention also provides extracts and isolatesof the same. As mentioned above, the Urodele ECM is loaded withantimicrobial peptides, growth promoting factors, collagen and laminins,and Urodele fractions of the ECM are useful according to the invention.

Extraction buffers are well known in the art. One such buffer is 4 Mguanidine and 2 M urea each prepared in 50 mM Tris-HCl, pH 7.4. Thepowder form of the ECM can be suspended in the relevant extractionbuffer (e.g., 25% w/v) containing phenylmethyl sulphonyl fluoride,N-ethylmaleimide, and benzamidine (protease inhibitors) each at 1 mM andvigorously stirred for 24 hours at 4° C. The extraction mixture can thenbe centrifuged and the supernatant collected. The insoluble material canbe washed in the extraction buffer, centrifuged, and the wash combinedwith the original supernatant. The supernatant can be dialyzed againstdeionized water. The dialysate can then be centrifuged to remove anyinsoluble material and the supernatant used immediately or lyophilizedfor long term storage. Such an isolate will contain growth factors inconcentrations specific to Urodeles.

In another aspect, the extraction is done by conditioning medium. Amethod of making Urodele tissue-specific extract by taking the powderedECM, forming a solution thereby generating a supernatant and aparticulate, wherein the supernatant is an extract and isolating theextract from the particulate. One also could grow cells on the ECM, andisolate the supernatant after a period of time of cell growth.

Synthetic Materials

Synthetic biocompatible and cyto-compatable material can be combinedwith the ECM, such as, for example, (a) a structural support for a sheetor a gel of the ECM, (b) a structural support for shaping the ECM, (c) acoating for the ECM (or a coating containing the particulate ECM), asupplemental gelling agent, or (d) a sustained release material for theparticulate ECM or an isolate thereof Such polymers have been known tobe applied to other ECM materials as a backing sheet, includingmaterials that are themselves biodegradable. Suitable synthetic materialfor a matrix can be biocompatible to preclude migration andimmunological complications, and can be able to support cell growth anddifferentiated cell function. Some are resorbable, allowing for acompletely natural tissue replacement. Some can be configurable into avariety of shapes and have sufficient strength to prevent collapse uponimplantation. Studies indicate that the biodegradable polyester polymersmade of polyglycolic acid fulfill all of these criteria (Vacanti, et al.J. Ped. Surg. 23:3-9 (1988); Cima, et al. Biotechnol. Bioeng. 38:145(1991); Vacanti, et al. Plast. Reconstr. Surg. 88:753-9 (1991)). Othersynthetic biodegradable support matrices include synthetic polymers suchas polyanhydrides, polyorthoesters, and polylactic acid. Furtherexamples of synthetic polymers and methods of incorporating or embeddingcells into these matrices are also known in the art. See e.g., U.S. Pat.Nos. 4,298,002 and 5,308,7

As a non-limiting example, the powder may be formulated with tri-blockco-polymers. See international pubished application WO2012131104 andWO2012131106, each of which is incorporated herein by reference in itsentirety. Other examples include poloxamers, which are nonionic triblockcopolymers composed of a central hydrophobic chain of polyoxypropylene(poly(propylene oxide)) flanked by two hydrophilic chains ofpolyoxyethylene (poly(ethylene oxide)). Poloxamers are also known by thetrade name Pluronics (BASF). Certain poloxamers are useful as sustainedrelease materials for pharmaceuticals.

Particles of the invention also may be encapsulated into a polymer,hydrogel and/or surgical sealant. As a non-limiting example, thepolymer, hydrogel or surgical sealant may be PLGA, ethylene vinylacetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua,Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgicalsealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.),TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.). Inanother embodiment, the particle may be encapsulated into any polymerknown in the art which may form a gel when injected into a subject. Asanother non-limiting example, the particle may be encapsulated into apolymer matrix which may be biodegradable. Additional examples ofpolymers for controlled release and/or targeted delivery may alsoinclude at least one controlled release coating. Controlled releasecoatings include, but are not limited to, OPADRY®,polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such asethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®.

Uses

The decellularized ECMs described herein are useful for growing cells,tissues, organs in virtually any in vivo, ex vivo, or in vitro use. TheECMs can be used as a substrate to facilitate the growth and/ordifferentiation of cells. In vitro, the ECMs are useful as a cell growthsubstrate to support the growth in culture of cells, including virtuallyany type of cells or cell-lines, including stem cells, progenitor cellsor differentiated cells. In one embodiment, the cells are cancer cells.In one embodiment, the cancer cells form nodules when grown on the ECMs.Cells on the substrate also may be grown into tissue, organ or body partprecursors, or even mature tissues or structures. Cells grown on ECMsmay be used for implantation, for wound dressings, for in vitro drugtesting, for modeling differentiation, etc. The cells may be matched intissue cell type to the ECM or unmatched. The cells are xenogenic.

The ECM of the invention is useful in vivo as a cell growth scaffold fortissue growth for any useful purpose, including repair, replacement oraugmentation of tissue in a subject in either humans or animals. Forexample, the materials are useful in repair and/or replacement of tissuelost or damaged during trauma or surgery, for example in loss of tissueafter tumor removal. The materials are useful for structural repair,such as inguinal hernia repair, parastomal reinforcement, soft tissuereinforcement, surgical staple-line reinforcement during, for example,bariatric surgery or lung resection, umbilical hernia grafts, Peyronie'srepair grafts, incision grafts and fistula plugs. The materials areuseful for wound dressings, such as for burns, graft and split-thicknessgraft coverings, ulcers including decubitis ulcers and dermal abrasionprocedures. The materials are useful for cosmetic purposes, for examplein breast, lip or buttock augmentation. An aspect of the inventionparticularly appealing for anti-adhesion surgical uses is the propertiesof the basement membrane, which inhibit or prevent adhesion. Thepresence of the AMPs make the ECM of the invention particularly wellsuited for the foregoing applications.

As mentioned above, the materials described herein can be molded orcontained within a structure to form desired shapes, such as, forcartilage repair or replacement by seeding the material with, e.g.,chondrocytes and/or chondroprogenitor cells. The materials can be groundinto a powder and used to reconstitute and/or form gels, as cell cultureadditives, as a powder, spray, liquid, suspension or coating forapplication to (a) a wound, (b) an implant, (c) a wound dressing, etc.

In one embodiment, for example, adipose stem cells are propagated in thecell growth scaffolds described herein. Adipose stem cells are ofmesodermal origin. They typically are pluripotent, and have the capacityto develop into mesodermal tissues, such as: mature adipose tissue;bone; heart, including, without limitation, pericardium, epicardium,epimyocardium, myocardium, pericardium, and valve tissue; dermalconnective tissue; hemangial tissues; muscle tissues; urogenitaltissues; pleural and peritoneal tissues; viscera; mesodermal glandulartissues; and stromal tissues. The cells not only can differentiate intomature (fully differentiated) cells, they also can differentiate into anappropriate precursor cell (for example and without limitation,preadipocytes, premyocytes, preosteocytes). Also, depending on theculture conditions, the cells can also exhibit developmental phenotypessuch as embryonic, fetal, hematopoetic, neurogenic, or neuralgiagenicdevelopmental phenotypes.

In one embodiment, a subject's own cells are dispersed within thematrix. For example, in the production of cartilaginous tissue,chondrocytes and/or chondroprogenitor cells can be dispersed within thematrix and optionally grown ex vivo prior to implantation. Likewise,skin cells of a subject can be dispersed within the scaffolding prior toimplantation on a damaged skin surface of a subject, such as a burn orabrasion.

When used as a gel, a non-limiting example is injecting the gel into asubject at a desirable site, such as in a wound. In one instance, thegel can be injected in a bone breakage or in a hole drilled in bone tofacilitate repair and/or adhesion of structures, such as replacementligaments, to the bone. In another use, finely comminuted particles canbe sprayed onto a surface of a subject, such as in the case of largesurface abrasions or burns. The scaffold can also be sprayed onto skinsutures to inhibit scarring. The ECM of the invention can be place orsutured in place inside the body at a surgical site such as mentionedabove. All of these treatments are embraced by the present invention.

Urodele decellularized ECM can be used also for sustained delivery oftherapeutic molecules, proteins or metabolites, to a site in a host.See, for example, U.S. 2004/0181240, which describes an amnioticmembrane covering for a tissue surface which may prevent adhesions,exclude bacteria or inhibit bacterial activity, or to promote healing orgrowth of tissue, and U.S. Pat. No. 4,361,552, which pertains to thepreparation of cross-linked amnion membranes and their use in methodsfor treating burns and wounds. The ECMs of the invention can be used inthe same manner.

Pharmaceutical Formulations

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.

The pharmaceutical compositions described herein may be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the step of bringing the active ingredient intoassociation with an excipient and/or one or more other accessoryingredients, and then, if necessary and/or desirable, dividing, shapingand/or packaging the product into a desired single- or multi-useconfiguration.

The ECM in accordance with the invention may be prepared, packaged,and/or sold in bulk, as a single unit dose, and/or as a plurality ofsingle unit doses. For example, the composition may comprise between0.1% and 100% (w/w) of the ECM. When other active agents are included,relative amounts of agents combined with the ECM of the invention willbe known to those of ordinary skill in the art, similar to those amountsused in combination with ECM as formulated in the prior art. Relativeamounts also may vary, depending upon the identity, size, and/orcondition of the subject being treated and further depending upon theroute by which the ECM is to be administered.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art. See Remington: THESCIENCE AND PRACTICE OF PHARMACY (21^(st) Ed.), A. R. Gennaro,Lippincott, Williams & Wilkins (Baltimore, Md., 2006); incorporatedherein by reference in its entirety.

EXAMPLES Example 1 Processing Axolotl Dermis

Axolotl dermis samples can be decellularized by preparing excisedsamples from healthy or healing axolotl dermal tissue and thensubjecting the samples to hypo/hyperosmotic soaks for cell lysis,solvent dehydration, and oven drying. Specific processing of thesegrafts includes storage in 15-26% NaCl, multiple hypo/hyperosmotic soaks(utilizing NaCl solutions and water), and then solvent dehydration usingethanol, and then evaporation of the solvent either with air drying oroven drying at 37° C.

Histological examination of native axolotl dermal tissue was performedto identify the presence of the notable ECM elements, such as thebasement membrane. See FIG. 1 and FIG. 4. Comparative histological andimmunohistochemical analysis of native axolotl dermal tissue and humanamniotic membrane was performed to compare the ECM structure andconstituents, and to assess relative concentration and distribution ofcritical constituents. See FIG. 2, FIG. 3, and FIG. 4. FIG. 3 showsimmunohistocemical staining via species-specific collagen IV and lamininantibodies of native axolotl dermal tissue and human amniotic membranetissue, at 40× magnification. FIG. 2 shows H&E and Alcian Blue staining(40×) of native axolotl dermal tissue and human amniotic membrane, andit demonstrates the comparable histoarchitecture and presence ofsulfated glycosaminoglycans in both tissues. Histological evaluationwith hematoxylin and eosin-stained, paired native and post-processedsections of axolotl dermal tissue (see FIG. 5) showed post-processpreservation of the extracellular matrix histoarchitecture and theabsence of cells or any significant concentration of cellular debris.

Example 2 Splitting and Lamination of Acellular Dehydrated AxolotlDermis

Decellularized dehydrated axolotl dermis can be split, via a mechanicalsplitter, to isolate heterogeneous matrix into homogenous sections.Isolated sections of desired thickness then can be rehydrated andlyophilized to obtain multilayered laminate structures of desiredorientation with facial surface features. More specifically, dual-sidedbasement membrane structure, with interior open porous matrix obtainedfrom the reticular dermis region of the dermal matrix, can beconstructed to obtain desired facial surface properties. Alternatively,isolated native section can be used in native form for desired clinicaloutcome. For example, open porous homogenous matrix of the reticulardermis can be used to obtain augmentation of soft tissue structures.

A laminated custom construct with sulfated gags on both facial surfaceand collagen IV and laminin could be obtained for desirabledual-surface, anti-adhesion and antimicrobial properties for clinicalbenefit. In addition, multilayer structures could be constructed toprolong in vivo durability of the graft.

Example 3 Preparation of Solubilized Acellular Dehydrated AxolotlDermis, Pericardium, Fascia Lata, Periosteum, Peritoneum, or Dura Mater

Decellularized dehydrated axolotl native or isolated section ofacellular urodele connectivue tissye matrix can be prepared bysectioning decellularized soft tissue structures into 1 cm² sections andhomogenizing the sections in a Warring blender (˜100 grams of tissue) inaqueous 1M glacier acetic acid for 30-60 seconds. Preparation of spongecan be obtained by the addition of varying volumes of water followedthen neutralization and lypoholization of the slurry in a mold ofdesired geometric shape. The resultant porosity will correlate to thevolume of water added to the matrix. Additionally, a selected range ofbioactive extracts can be added to the slurry prior to neutralization,including particulated or small protein constituents extracted fromdigested human or urodele mineralized and nonmineralized connectivetissues, such as demineralized bone matrix, elastin, or bone morphogenicproteins, which can be covalently loaded into constructs. Extracts willbe covalently bound with collagen fibers after neutralization and returnto physiological condition where fibrillogenisis will occur. Subsequentrelease of bioactive constituents will occur during proteolycticdegradation in vivo and ensure molecules are not consume or exposedduring acute inflammation in vivo. Alternatively, aqueous NaCl can beadded to the slurry, prior to neutralization, to obtain a sustained, lowviscosity solution for injection, which is stable at room temperature.Injection of slurry through ion-selective membrane will remove salt ionsand permit for fibrillogenisis to occur post injection and formation ofthree-dimensional matrix.

Example 4 Preparation of Sterilized Particulated or Powder Form ofMineralized and Non-Mineralized Decellularized and Dehydrated UrodeleConnective Tissue Matrix

Following decellularization of sections of mineralized collagen urodeleconnective tissue, one can perform a demineralization process, similarto that employed by Urist, and solvent or lyophilization dehydration,cryomilling of sectioned acellular demineralized, mineralized, ornon-mineralized urodele connective tissue extracellular matrix, therebyto obtain particulated or powder form of the ECM with preservedhistoarhictiecutre and function. The final particle size distributioncan be varied depending on duration and sieving, post-cyromilling,between 125 and 850 microns. Low-dose cold gamma irradiation or e-beamirradiation (<25 Kgy) can be employed to sterilize acellular ECM sheets,particulate or powder and custom engineered constructs

Example 5 In Vitro Characterization of Acellular Mineralized andNon-Mineralized Urodele Healthy or Healing Connective Tissue Matrix

Through a series of in vitro analyses one can verify decellularizationand preservation of native or custom engineered functional andstructural properties of decellularized extracellular matrix constructsand/or particulate, including multilayer laminated constructs such as adual-sided basement membrane sheet matrix or isolated native homogenousopen porous matrix or solubilized, lyophilized and loaded ECM-derivedcollagen sponge. DNA content, as a marker for cell debris, can beemployed to assess decellularization quantitatively, using a single,ethanol-based extraction technique with a fluorometric dye, Quant-iTPicoGreen (Molecular Probes, USA), in a ratio of 170 μl working solutionto 30 μl samples/standards in a 96-well plate. Paired native andpost-processed analysis and comparison to commercially available tissueECM's can be performed to verify acceptability. See FIG. 6.

ELISA analysis for quantification of bioactive constituent and nativeand post process protelyctic resorption profiles can be performed. Upondigestion with collagenase (232-262 mg/unit activity),normalized-weight-to-surface-area sections of decellularized anddehydrated urodele ECM tissue or constructs (Sigma, USA), in a pH 7.6buffer (50 mM Tris-HCl, 200 mM CaCl₂, 50 mM NaCl) for 24 hours at 37°C., can be analyzed at various time points to construct a relativeresorption curve of pre- and post-process tissue to verify preservationof histoarchitecture. Solubilized collagen following digestion can beassessed, using a Sircol kit (Biocolor Ltd., UK), in 100 μl aliquots ofacid/salt-washed digests. Specifically, levels of BMP 2/4 and TGF-1growth factors or sulfated gags can be assessed, following digestion, bymeans of commercially available ELISA kits (R&D Systems, Minneapolis,Minn.). Protein content in 1:10 dilution digests can be measured via astandard Bradford absorbance assay.

Microscopic evaluation of samples can be performed using fixation in 4%paraformaldehyde and paraffin embedding, sectioning at 5μm, and routinehistological staining (Histoserv, Inc., USA). Longitudinal crosssections can be stained with hematoxylin and eosin. Images can beacquired and anyzed using standard brightfield techniques on an OlympusIM inverted microscope. Samples can analyzed using scanning electronmicroscopy after dehydration in a graded ethanol series (15%, 30%, 50%,70%, 95%, and 100%), critical-point drying in CO₂, and sputter coatingwith gold. Samples can be visualized in an FEI Quanta 600 FEG scanningelectron microscope, and representative images of scaffoldultrastructure can be acquired.

Direct cell contact methodology (ISO10993, Part 5), for qualitative cellviability assessment at 24 hours, can be conducted for cytotoxicitytesting and at extended time points (48 and 72 hours) to gauge cellproliferation and adhesion efficiency. A manual count of non-adherentcells in a hemocytometer, following transfer and trypsinization of theculture wells, can be conducted. A CellTiter 96 assay can performed toquantify viable cells after four days. A live/dead cell staining kit canused as well to visualize scaffolds via fluorescence microscopy at 24hours and day four, thereby to verify biocompatibility.

1-33. (canceled)
 34. A composition comprising decellularized Urodeleextracellular matrix (ECM) having functional activity.
 35. Thecomposition of claim 34, wherein the decellularized Urodele ECM isobtained from a salamander from family Ambystomatidae, Cryptobranchidae,Amphiumidae, Proteidae, or Sirenidae.
 36. The composition of claim 34,wherein the decellularized Urodele ECM is obtained from neotenicUrodele.
 37. The composition of claim 34, wherein the decellularizedUrodele ECM has reduced or no antigenicity and/or immunogenicity ascompared to a naturally occurring Urodele ECM.
 38. The composition ofclaim 34, wherein the composition further comprises devitalized Urodeleepithelial cells.
 39. The composition of claim 34, wherein thedecellularized Urodele ECM is derived from a skin of a Urodele.
 40. Thecomposition of claim 34, wherein the decellularized Urodele ECM isderived from an axolotl.
 41. The composition of claim 34, wherein thecomposition is a pharmaceutical or cosmetic composition.
 42. Thepharmaceutical or cosmetic composition of claim 41, wherein thepharmaceutical or cosmetic composition further comprises an agent. 43.The pharmaceutical or cosmetic composition of claim 42, wherein theagent is xenogenic to Urodele.
 44. The pharmaceutical composition ofclaim 42, wherein the pharmaceutical composition further comprises apharmaceutically acceptable excipient.
 45. The pharmaceuticalcomposition of claim 44, wherein the agent is a therapeutic agent. 46.The pharmaceutical composition of claim 45, wherein the therapeuticagent is an antimicrobial agent, an anti-inflammatory agent, an agentthat promotes wound healing, or an agent that promotes tissueregeneration.
 47. The pharmaceutical composition of claim 46, whereinthe pharmaceutical composition further comprises a carrier.
 48. Thepharmaceutical composition of claim 42, wherein the agent is a growthfactor, a cytokine, a chemokine, a retinoid, a steroid, or an emollient.49. The pharmaceutical or cosmetic composition of claim 41, wherein thepharmaceutical or cosmetic composition is in a form of a powder, a gel,or a paste.
 50. The pharmaceutical or cosmetic composition of claim 41,wherein the pharmaceutical or cosmetic composition further comprisessynthetic biocompatible material and/or synthetic cyto-compatiblematerial.
 51. The pharmaceutical or cosmetic composition of claim 50,wherein the synthetic biocompatible material and/or syntheticcyto-compatible material provide sustained release of decellularizedUrodele ECM.
 52. The pharmaceutical or cosmetic composition of claim 51,wherein the synthetic biocompatible material and/or syntheticcyto-compatible material comprise tri-block co-polymers or poloxamers.